The Toxoplasma gondii Genome Project

Sequencing Methodology

TIGR is sequencing the Toxoplasma gondii B7 strain (a recent clonal isolate of the type II parasites most commonly associated with AIDS). The sequencing methodology utilized at TIGR is a random shotgun sequencing strategy consisting of the following steps:

a. Preparation of random genomic shotgun libraries of T. gondii DNA in small (2.0 kb) and medium (10 kb) insert plasmid vectors, and a random genomic shotgun large insert BAC library (80-100 kb).

b. Sequencing both ends of randomly selected clones from both the small and medium insert plasmid libraries to provide at least 3X coverage of the genome (375,000 sequences). Sequencing both ends of 8000 randomly selected clones from the large insert BAC library to provide a scaffolding for linking of assemblies and contigs during closure of the genome.

c. Assemble the genomic sequence from the collection of random sequence fragments. Note that the use of various size insert libraries provides linking information that will facilitate the ordering of many unfinished contigs, enabling gap closure in individual laboratories whenever warranted.